The three dimensional structure of engineered trypsin molecules will be determined at high resolution. The design of mutant trysins will include substitutions, deletions and insertions which are designed to test the role of specific amino acids in promoting catalysis and in defining substrate specificity. Alterations in the structure of trypsin are designed by modeling these changes using computer graphics and by considering current hypotheses about catalysis in serine proteases. These proteins will be constructed using site specific mutagenesis and will then be expressed in E. coli. This will produce sufficient material for protein structure determination using x-ray crystallography. Three dimensional structures will be determined both in the presence and the absence of inhibitors and substrate analogs to further characterize the role of each altered amino acid. This analysis will help resolve long-standing questions about how enzymes use favorable binding energy to greatly increase reaction rates. Two of the many specific experiments that are outlined in this proposal test the roles of Asp 102 (part of the catalytic triad) and Gly 226 (involved in both the trypsinogen - trypsin transition and substrate specificity). The carboxylic acid side chain of Asp 102 will be replaced with a glycine and the carboxylic acid will come from substituting Ser 214 with a Glu. Although these residues are distant in the sequence, they are close enough in the three dimensional structure that Glu 214 may substitute in the catalytic triad. The role of Gly 226 in the trypsinogen to trypsin transition will be tested by replacing it with alanine. In trypsin, Gly 226 adopts a main chain conformation that is inaccesible to all other amino acids. Substitution with Ala at position 226 should favor the trypsinogen-like conformation over that of trypsin. Solving the three dimensional structure of Ala 226 trypsin will determine if Gly at this position is requied for the enzyme to adopt the active conformation.